The volume of crime committed in the United States has risen with the increase of population and expansion of population centers. A large portion of violent crimes involve the creation of body fluid evidence having the potential of providing significant information about the perpetrator of a particular offense. Although the forensic science community has made tremendous effort in using this evidence, the results have historically been limited and are not useful in many situations when dealing with human remains and crime scene evidence. Identification by genetically inherited markers has long been seen as a possibility that would overcome most of the problems that are encountered when identification is not accomplished by fingerprints, forensic odontology, medical records or other methods. The establishment of a genetically inherited method that could be used for identification would have tremendous impact on investigation of the violent crimes of sexual assault and murder, identification of human remains and missing persons, and disputed parentage.
Methods enabling the matching of unidentified tissue samples to specific individuals would have wide application in the criminal justice system and the forensic sciences. With the possible exception of monozygotic twins, each individual in the human population has a unique genetic composition which could be used to specifically identify each individual. This phenomenon presents the theoretical possibility of using DNA sequence variation to determine whether a forensic sample was derived from any given individual.
Genetic marker systems, including blood groups and isoenzymes, have been used by forensic and medical serologists to provide estimates of individuality ranging from 1:1000 to 1:1,000,000 using 10 to 15 markers. Numbers in this range are often not available since a large percentage of the evidence does not yield results for ten genetic marker systems. Forensic scientists, investigators and the court system have been using inclusions as low as 1:5 to 1:100 in a population to bolster their case against defendants.
The fields of forensic and medical serology, paternity testing, and tissue and sample origin has been altered by the use of DNA sequence variation, e.g., satellite sequences and variable number of tandem repeats (VNTRS) or AMP-FLPS, in the crime laboratory, the court, hospitals and research and testing labs. Inclusion probabilities stated by the laboratories performing the analyses in such cases often exceed 1:1,000,000. The first implementation of DNA typing in forensics, was Jeffreys' use of a multilocus DNA probe "fingerprint" that identified a suspect in a murder case occurring in England. In the United States, DNA profiling has been established using a battery of unlinked highly polymorphic single locus VNTR probes. The use of these batteries of probes permits the development of a composite DNA profile for an individual. These profiles can be compared to ethnic databases using the principles of Hardy-Weinberg to determine the probability of the match between suspect and unknown forensic samples.
The application of VNTRs to gene mapping, population genetics, and personal identification has been limited by the low frequency and asymmetric distribution of these repeats in the genome and by the inability to precisely determine alleles with Southern hybridization-based detection schemes. The inability to make precise allele determinations complicates the application of VNTRs to personal identification. Binning protocols have been devised in which all alleles occurring within a region of the gel are treated as the same allele for genotype calculations. Since the allele distribution appears continuous because of the limited resolving power of Southern gels, heterozygotes with alleles of similar size may be scored as homozygotes. These features have led to claims that VNTR loci are not in Hardy-Weinberg equilibrium, and therefore the method for calculating the significance of a match is not agreed upon.
Although these methods have markedly improved the power of the forensic and medical scientist to distinguish between individuals, they suffer from a number of shortcomings including a lack of sensitivity, the absence of internal controls, expense, time intensity, relatively large sample size, an inability to perform precise allele identification and problems with identifying degraded DNA samples.
Medical and forensic studies have also employed the polymerase chain reaction (PCR) to examine variation in the HLA locus. PCR has also been used to amplify short VNTRs or AMP-FLPs. The use of PCR addresses some of the problems of sensitivity and sample degradation, however, the HLA typing system, still has some problems. A simpler, more powerful technique is needed which makes use of the most recent advances in DNA technology.
The present invention involves the novel application of these advances to medical and forensic science. In the present invention novel classes of highly polymorphic, primarily trimeric and tetrameric, short or simple tandem repeats (STRs) which are present within the human genome have been identified. These STRs have characteristics suitable for inclusion in a DNA profiling assay. This assay incorporates internal or external standards, provides higher sensitivity, requires shorter analysis time, lowers expense, and enables precise identification of alleles. The STRs are amplified with great fidelity and the allele patterns are easily interpreted. Amplification of highly polymorphic tandemly reiterated sequences may be the most cost effective and powerful method available to the medical and forensic community.
The DNA profiling assay of the present invention has features which represent significant improvements over existing technology and brings increased power and precision to DNA profiling for criminal justice, paternity testing, and other forensic and medical uses.